This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Objective: To study a new vaccine approach to HIV. Immune epitope dominance results in one or a few epitopes comprising the greatest part of the immune response to SIV infection and vaccination. We know that some of these dominant epitopes are not useful in controlling SIV because they either mutate rapidly (Mamu-A*01 Tat SL8) or simply do not control viremia on their own (Mamu-A*01 Gag CM9). It may be that subdominant immune responses are more efficacious than dominant immune responses, but are at low frequency. Therefore, we have designed a series of Minigenes which are regions of SIV proteins that encode no more than one immunodominant epitope. By separating immunodominant epitopes from subdominant epitopes, we hope to allow increased frequency of the subdominant epitopes resulting in greater breadth of the immune response. In addition, while we have had successful vaccine trials in rhesus macaques with DNA/Ad5 as vaccine vectors, adenovirus is now less favored as a vaccine vector in humans due to widespread prior infection with adenovirus and due to the poor results obtained in the recent Merck STEP trial using this vector. Therefore, at the same time that we are exploring the minigene concept, we are also exploring the use of alternate vectors. We will put minigenes into rBCG, rShigella, rYF-17D and possibly other vectors, vaccinate macaques and challenge them to assess the immunogenicity of these new approaches. SUBPROJECT PROGRESS: We have completed our studies of viral protein/epitope presentation. We found that there is a clear hierarchy of presentation, which we can use to enhance vaccine development by targeting those proteins/epitopes that are presented early. In addition, we found that not all parts of a single protein are presented at the same time. Therefore, it would be useful to present earlier proteins or parts of proteins in our minigene constructs. Production of the mini-gene vaccine constructs for Gag, Nef, Rev, and Tat in rBCG is complete and we have vaccinated these animals with an intradermal dose, and two oral doses of this vaccine. We have seen disappointingly few responses to the SIV sequences encoded this vaccine, although we know that robust immune responses were developed against the BCG portion of the vector. The rShigella vaccine is still under production by our collaborators. Some animals that were vaccinated with rBCG, were subsequently vaccinated either with rYF-17D encoding most of the Gag protein, or rYF-17D encoding minigenes of Gag, Vif and Nef. In the first case, we saw only weak responses, but upon low dose mucosal challenge with SIVsmE660, we saw robust expansion of epitopes in proteins in the vaccine. In one case, the animal had 40% of CD8+ T cells that were responding to Gag CM9 presented in the context of Mamu-A*01. Unforunately, this was not protective and the animal sustained high viral loads. The vaccination with rYF-17D encoding minigenes was less robust. It is possible that the site in which we inserted the SIV sequences into the rYF-17D is not ideal, and we are exploring other potential sites. This research used WNPRC Animal Services, Genetics Services, and Immunology &Virology Services. PUBLICATIONS: Sacha JB, Giraldo-Vela JP, Buechler MB, Martins MA, Maness NJ, Chung C, Wallace LT, Le[unreadable]n EJ, Friedrich TC, Wilson NA, Hiraoka A, Watkins DI. Gag- and Nef-specific CD4+ T cells recognize and inhibit SIV replication in infected macrophages early after infection. Proc Natl Acad Sci U S A. 2009 Jun 16;106(24):9791-6. Epub 2009 May 28. PMID: 19478057 Bonaldo MC, Martins MA, Rudersdorf R, Mudd PA, Sacha JB, Piaskowski SM, Costa Neves PC, Veloso de Santana MG, Vojnov L, Capuano S 3rd, Rakasz EG, Wilson NA, Fulkerson J, Sadoff JC, Watkins DI, Galler R. Recombinant Yellow Fever Vaccine Virus 17D Expressing SIVmac239 Gag Induces SIV-Specific CD8+ T Cell Responses in Rhesus Macaques. J Virol. 2010 Jan 20. [Epub ahead of print] PMID: 20089645